This invention relates generallly to biochemical assays, and more specifically, to means for detecting protein.
The detection of proteins has important application in biochemistry, clinical chemistry and medicine. For example, determing the presence and quantity of proteins in urine or blood is diagnostically important and may be of crucial importance in evaluating an emergency protocol whee proteinuria is suspected, which may indicate such conditions as heart failure or the onset of renal disease. Moreover, in conventional biochemical assays such as electrophoresis and chromatography, which are used for separating organic molecules, the detection of general proteins is commonly required.
In order to visually detect the presence of protein, a multi-step process is presently necesssary using conventional techniques. A solution suspected of containing protein is placed on a substrate as to which any protein will attach or become engulfed. A stain which preferentially binds with protein is then applied to the substrate and allowed to react with any protein present. Unbound stain is removed and the substrate evaluated for stain, indicating the presence of protein.
Presently, the preferred stain for general protein is coomassie blue, or anazolene sodium. This stain has a sulfonate group which binds ionically to the positively charged amines of protein. The dye takes an average of six to twelve hours to stain, for example, a polyacrylamide electrophoretic gel. Such a lengthly time period significantly decreases the utility of the assay. Recently, a new stain based on the chemistry of nickel has been developed (Kodavue Electrophoresis Kit, Eastman Kodak Co., Rochester, N.Y.). While the time involved is significantly reduced, the assay requires a cumbersome number of fixing, washing and drying steps.
Because traditional stains are visible both before and after attachment to the proteins, standard protein assays cannot be used to detect protein in solution without first immobilizing the protein. The presence of protein is determined solely by the affinity of the stain for the protein relative to the washing solution.
It will therefore be appreciated that there exists a long standing need for a stain for protein which is fast, effiient and easy to use. Additionally, in order to allow use of the stain to detect protein which is free in solution, the dye should exhibit a visually detectable change upon binding to protein. The present invention satisfies these needs and provides related advantages as well.